EP0563858B1 - Verfahren zur Bestimmung der Anzahl lebender Mikroorganismen - Google Patents

Verfahren zur Bestimmung der Anzahl lebender Mikroorganismen Download PDF

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Publication number
EP0563858B1
EP0563858B1 EP93105182A EP93105182A EP0563858B1 EP 0563858 B1 EP0563858 B1 EP 0563858B1 EP 93105182 A EP93105182 A EP 93105182A EP 93105182 A EP93105182 A EP 93105182A EP 0563858 B1 EP0563858 B1 EP 0563858B1
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EP
European Patent Office
Prior art keywords
viable count
membrane
determining
luminescence
count according
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Expired - Lifetime
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EP93105182A
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English (en)
French (fr)
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EP0563858A1 (de
Inventor
Masaya Kawakami
Susumu Seto
Seiken Tei
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Merck Ltd Japan
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Nihon Millipore KK
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Priority claimed from JP02342093A external-priority patent/JP3208892B2/ja
Application filed by Nihon Millipore KK filed Critical Nihon Millipore KK
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property

Definitions

  • the present invention relates to a method of determining a viable count. More paricularly, the invention relates to a rapid, convenient, and highly sensitive method of determining a viable count by detecting adenosine triphosphate (ATP) contained in microbes, which probably exist in industrial water, raw materials, intermediates, and products used in the industries of foods, pharmacy, cosmetics, electronics, etc.
  • ATP adenosine triphosphate
  • the measuring vessel must contain viable microbes in a number above the lower limit of detection for the luminometer (for example, ⁇ 1 x 10 3 or 1 x 10 4 cell/ml), thus requiring, particularly for a sample solution having extremely low viable count, a large volume of sample must be filtered.
  • the present inventors have once developed a filter comprising a number of small hydrophilic filtration membrane sections substantially completely isolated from each other with hydrophobic partitions, and invented a method comprising adding reagents for extraction and luminescence induction to the microbes collected on said filtration membranes by spraying, and treating the obtained preparation with a high-sensitivity bioluminescent image analysis system, to submit an application (Japanese Patent Application No. 3-40615; and PCT-JP92-00145).
  • the present invention provides a method of determining a viable count in a sample characterized in that it comprises:
  • a filter 1 used in performing the method according to the present invention comprises a number of small hydrophilic filtration membrane sections 2 substantially completely isolated from each other with hydrophobic partitions 3.
  • This filter is made from a filter membrane 4 and hydrophobic partitions 3 by printing of a hydrophobic ink pattern on the membrane. The ink may penetrate into the filter element and the partitions may be flush with or higher than the surface of the membrane.
  • the ATP extracting reagent suitable for use in the present invention includes alcohols, ethers, esters, and halogenized derivatives of methane, ethane, methylene or ethylene, as well as acetonitrile, triethylamine and others having boiling point below 120° C. Methanol and ethanol are particularly preferred.
  • Extracting effect is further enhanced by admixing 0.1 to 5 % by weight of hydrochloric acid, organic acids having b.p. below 120 ° C, or basic compounds such as ammonium hydroxide, organic amines and others having b.p. below 110 ° C with the above alcohols.
  • hydrochloric acid and organic acids having a tendency of volatility significantly enhance the extracting effect, they reduce the activity of the luminescence-inducing enzyme, when left in the sample even in a trace amount, thus necessitating more complete evaporation, and sometimes further spray of a neutralizing reagent to offset the acidity.
  • Ammonium hydroxide is, therefore, particularly preferable, which has high volatility and does not inhibit the induction of luminescence.
  • ATP extracting reagents may be evaporated off at a temperature between ambient temperature and 80° C, preferably between 40° C and 70° C.
  • the use of a fine spray of said extracting reagents with an ultrasonic type sprayer (such as manufactured by Matsushita Electric Industries Co., Ltd.) is convenient and effective to add an exact amount required.
  • the membrane filter to be used in the method according to the present invention is a film or sheet of a hydrophilic filtration membrane, precisely made of plastic materials, such as hydrophilic polytetrafluoro-ethylene, hydrophilic poly(vinylidene fluoride), hydrophilic polysulfone, hydrophilic polycarbonate, hydrophilic polyamide, hydrophilic polyethylene and hydrophilic polypropylene, and cellulosic material, such as acetylcellulose and nitrocellulose, and having a number of uniform micropores with pore size from 0.1 to 1 ⁇ m.
  • plastic materials such as hydrophilic polytetrafluoro-ethylene, hydrophilic poly(vinylidene fluoride), hydrophilic polysulfone, hydrophilic polycarbonate, hydrophilic polyamide, hydrophilic polyethylene and hydrophilic polypropylene, and cellulosic material, such as acetylcellulose and nitrocellulose, and having a number of uniform micropores with pore size from 0.1 to
  • a filter cup equipped with a filtration membrane with a pore size from 0.1 to 1 ⁇ m, preferably from 0.2 to 0.45 ⁇ m at the bottom of a cylinder (diameter: 10 - 40 mm ⁇ ) made of plastics, as described in Japanese Patent Application No. 2-253093.
  • a sample solution is filtered using a filtration device, which is a cup-shaped vessel to be filled with the solution, equipped with the above membrane filter to entrap viable microbes thereon.
  • the membrane filter is then subjected to a fine spray with the aforementioned ATP extracting reagent for the time period from 5 seconds to around 5 minutes, using such a sprayer as of ultrasonic type manufactured by Matsushita Electr. Ind. Co., Ltd. to extract microbial ATP, at a temperature from ambient to 80° C, preferably from 40 ° C to 70 ° C.
  • Proper time period for spray may vary depending on the species of microbes to be tested and the sort of extracting reagent to be used.
  • Heating may be applied during and/or after spray, but in a preferred manner, the membrane filter is subjected to the fine spray at ambient temperature, and then heated to a temperature from ambient to 80° C, preferably from 40° to 70° C to rapidly evaporate the residual extracting reagent off.
  • a luminescence inducing reagent of luciferin/luciferase type is added over the membrane filter.
  • the reagent may be added onto the filter cup with a pipette, or with a usual sprayer, to induce luminescence. Otherwise (less than 100 cells/membrane), since a more precise spray in small amounts is required, spraying will be carried out with an ultrasonic type sprayer and the like.
  • an increased concentration of the luminescence-inducing reagent of luciferin/luciferase type is used when inducing luminescence by spraying it with a sprayer, or by adding it with a pipette (in the case where such a large count as above 100 cells/membrane is expected).
  • the reason is that the increased concentration will cause an increased rate of luminescence reaction, resulting in an increased quantity of luminescence.
  • This is a more effective way for samples with particularly low count (i.e., less than 100 cells/ membrane, particularly less than 50 cells/membrane), which should be determined with a bioluminescence image anylysis system.
  • the concentration of luminescence inducing reagent to be used is preferably increased to a range from twice to ten times, particularly from three to six times as high as standard concentration.
  • the rate of luminescence is accelerated to enhance the level of luminescence of bright spots, at the same time accelerating the speed of detection.
  • standard concentration means the concentration as specified for regular use of conventional luminescence-inducing reagents (for example, a luciferin/luciferase type reagent Lucifer LUTM commercially available from Kikkoman Co., Ltd.)
  • luminescence inducing reagent as manufactured by Kikkoman Co., Ltd. (Lucifer LUTM ), which is normally used in a diluted form of 70 mg of the dry reagent diluted with 5 ml of water, is used in a concentration from about three to six times higher than said concentration for regular use, as stated above, the time for emission of light after the spray of the reagent can be reduced, and the quantity of luminescence can be increased almost directly in proportion to its concentration.
  • the membrane filter thus allowed to emit light can be subjected to counting, using a luminometer, such as Luminescence Reader BLR-201TM (an improved model), Aloka Corporation, or to imaging bright spots appearing on the filter, using any devices for bioluminescence image analysis system, such as ARGUS-50/CLTM (provided with a tapered fiber), Hamamatsu Photonics, Co., Ltd. to determine the viable count.
  • a luminometer such as Luminescence Reader BLR-201TM (an improved model), Aloka Corporation
  • bioluminescence image analysis system such as ARGUS-50/CLTM (provided with a tapered fiber), Hamamatsu Photonics, Co., Ltd. to determine the viable count.
  • ARGUS-50/CLTM provided with a tapered fiber
  • Hamamatsu Photonics Co., Ltd.
  • the outline of the system is as shown in Fig. 3, in which (A) is an entire outline and (B) is an enlarged partial view.
  • This system comprises a preparation holder 9 for supporting the membrane filter (preparation) 5 after treatment with the aforementioned extracting and luminescence-inducing reagents; a total reflection plate 6; a shading housing 8; tapered fibers 7, which are juxtaposed to said membrane fileter as closely as possible to detect luminescence in a two-dimensional extent; an ultrahigh-sensitivity television camera 10 consisting of a photoamplifying component and a camera tube; a camera controller 11; an image processor 12; a data analysis device 13; and a monitor television 14.
  • ARGUS-50/CLTM of tapered fiber input type, Hamamatsu Photonics, Co., Ltd., or those having a similar counting performance is particularly preferred.
  • a cooled solid-state camera device may be employed, wherein it is cooled to a temperature from about -30° C to -120 ° C to restrain noises from the camera itself so that it can accumulate even very faint light.
  • a cooled CCD digital imaging system is available from Hamamatsu Photonics.
  • the system may be operated by placing the tapered fibers 7 in the camera tube portion and the ultrahigh-sensitivity television camera underneath the preparation holder and placing the preparation holder containing a preparation on the tapered fibers.
  • preparation 5 is placed as closely as possible to tapered fibers 7, thereby to significantly enhance the measurement sensitivity.
  • sprayer(s) for extracting and luminescence-inducing reagents, and other associated equipments such as preparation carrier may be set in a combined format.
  • viable microbes are entrapped on a membrane filter in an advantageous form as hitherto stated, and sujected to the microbial ATP extraction in situ, using a fine spray of a preferred volatile extracting agent. Subsequently, this solvent is rapidly evaporated off by subjecting the filter to a temperature from ambient to 80 ° C, preferably from 40 ° C to 70 ° C, before a direct spray of luminescence-inducing reagent to induce luminescence, which is subjected to a measurement using a high-sensitivity bioluminescence image analysis system to determine the viable count.
  • a membrane filter comprising a number of small hydrophilic sections completely isolated from each other with hydrophobic partitions
  • all the viable microbes are well dispersed and entrapped within the small sections.
  • the extracting solution solution of ATP extracting reagent
  • the solution of luminescence-inducing reagent luciferin/luciferase type
  • luciferin/luciferase type both to be sprayed thereon are designed so as to keep remaining within the sections without diffusing outside from each section nor being diluted. This allows the microbial constituent that has become emitting light to be retained closely at the location of the microbe in a relatively high concentration, so that the above mentioned effectiveness are further augmented, enabling an extremely sensitive determination to be attained.
  • any noise luminescence originated from other than viable microbes can readily be erased, so that even an extremely low viable count (e.g., several cells/membrane, can be determined automatically, as well as rapidly and conveniently.
  • a fine spray of 90% ethanol was then applied to the membrane for 30 seconds, as in Example 1, to extract ATP, and, after evaporating the solvent off at a temperature from 50° C to 60 ° C, a fine spray of a luminescence-inducing reagent (Lucifer-LUTM, Kikkoman Co., Ltd.) was applied thereto for 10 seconds to induce luminescence.
  • a luminescence-inducing reagent Lucinescence-inducing reagent
  • the preparation was loaded to an image analyzer (ARGUS-50/CLTM, Hamamatsu Photonics Co., Ltd.), and, after accumulating photons for two minutes and processing the image, only bright spots having luminance above the threshold value were imaged to determine the viable count.
  • the results obtained are shown in Table 4, along with the results from determining the viable count for the same sample solution by means of standard agar plate method using a potatoe dextrose/agar medium cultured at 30° C for 48 hours, for comparison.
  • Table 4 Standard Agar Plate (CFU/1 ml sample soln.) Present Invention (No. of bright spot/membrane) 23 20 26 21 21 20 23 19 20 17
  • a sample solution containing about 30 cells/ml of Saccharomyces cerevisiae (IFO 0209) was prepared by culturing and diluting according to Example 4. 1 ml of this solution was filtered through a filtration membrane as used in Example 4, and, after collecting microbes, a fine spray of methanol containing 3% by weight of triethylamine was applied to the membrane for 30 seconds, to extract ATP.
  • Example 4 After leaving it at ambient temperature for three minutes, then inducing luminescence by spraying the luminescence-inducing reagent for 10 seconds, and evaporating solvent off at a temperature from 50 ° C to 60 ° C, as in Example 4, the preparation was loaded to an image analyzer to accumulate photons for two minutes, and only bright spots having luminance above the threshold value and originated from viable microbes were imaged to determine the viable count. The results obtained are shown in Table 6. Also, the results obtained from the same sample solution by means of standard agar plate method using a pepton dextrose/agar medium cultured at 30° C for 48 hours are shown in Table 6.
  • a sample solution containing about 1 x 10 3 cells/ml of Staphylococcus aureus (IFO 3060) was prepared by culturing and diluting as in Example 5. After filtering 5 ml of this solution and collecting microbes on the membrane as in Example 5, ATP was extracted as in Example 1, using 90% ethanol containing 1% of ammonium hydroxide (aqueous ammonia) instead of 90% ethanol, then luminescence was induced by spraying a luminescence-inducing reagent (Lumit-PMTM, Lumac Co.) for 20 seconds, and the quantity of luminescence was measured.
  • a luminescence-inducing reagent Liit-PMTM, Lumac Co.
  • a diluted solution of Saccharomyces cerevisiae (IFO 0209) containing about 100 cells/ml was prepared as in Example 1. 0.2 ml of this solution was filtered through a uniquely latticed hydrophilic filtration membrane (47 mm ⁇ , Nippon Millipore Limited) and dried. Extracting reagent was then sprayed for 30 seconds as in Example 1 to extract ATP.
  • the present invention is advantageous in that it provides a method of determining a viable count using the techniques of the ATP bioluminescence method, wherein the use of particular volatile extracting reagents and an evaporating process allows ATP to be extracted effieciently from viable microbes entrapped and collected on a membrane filter, and prevents any inhibition of enzyme (i.e., luciferase) activity and adverse effect of ATP degeneration enzymes during subsequent luminescence-inducing process.
  • enzyme i.e., luciferase
  • the present invention is also advantageous in that, in the present method, the use of a membrane filter comprising small sections isolated from each other with slightly protruded partitions, and a spray process for adding reagents, combined with an improved bioluminescence image analysis system, which makes a highly sensitive and rapid detection of even faint luminescence possible, allows a viable count to be determined in a substantially reduced time, as compared with conventional methods, and further enables to carry out a highly sensitive, rapid, and convenient determination for samples having a relatively small count, determination of which has formerly been difficult.

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Claims (11)

  1. Verfahren zur Bestimmung der Anzahl lebensfähiger Mikroben in einer Probe, gekennzeichnet durch:
    Filtrieren einer Lösung der (betreffenden) Probe durch ein hydrophiles Membranfilter zum Einfangen darin enthaltener lebensfähiger Mikroben auf einer Filtrationsmembran dieses Filters;
    Aufsprühen eines flüchtigen Extraktionsreagenz eines Kp unter 120°C auf die Membran zur Extraktion von mikrobiellem Adenosintriphosphat;
    Anschließendes oder gleichzeitiges Einwirkenlassen von Umgebungstemperatur oder erhöhter Temperatur auf die Membran zur Verdampfung flüssiger Bestandteile;
    Aufsprühen einer Lösung eines lumineszenzinduzierenden Reagenz vom Luciferin/Luciferase-Typ auf die Membran zur Induktion von Lumineszenz und
    Bestimmen der Lumineszenzmenge unter Verwendung einer für eine solche Messung geeigneten Vorrichtung.
  2. Verfahren zur Bestimmung der Anzahl lebenfähiger Mikroben nach Anspruch 1, wobei das hydrophile Membranfilter eine Anzahl kleiner hydrophiler Filtrationsmembranabschnitte, die voneinander durch hydrophobe Trenneinrichtungen praktisch vollständig isoliert sind, aufweist.
  3. Verfahren zur Bestimmung der Anzahl lebensfähiger Mikroben nach Anspruch 1 oder 2, wobei es sich bei dem hydrophilen Membranfilter um einen am unteren Ende bzw. Boden eines Kunststoffzylinders mit einer hydrophilen Filtrationsmembran ausgestatteten Filterbecher handelt.
  4. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei das hydrophile Membranfilter aus einem Werkstoff, ausgewählt aus der Gruppe hydrophiler Formen von Polytetrafluorethylen, Poly(vinylidenfluorid), Polycarbonat, Polyamid, Polysulfon, Polyethylen, Polypropylen und Mischungen und Mischpolymeren hiervon, Acetylcellulose und Nitrocellulose, hergestellt ist.
  5. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei das flüchtige Extraktionsreagenz für ATP aus Methanol oder Ethanol besteht.
  6. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei das flüchtige Extraktionsreagenz für ATP aus einem Gemisch aus Methanol oder Ethanol mit 0,1 bis 5 Gew.% Ammoniumhydroxid besteht.
  7. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei das lumineszenzinduzierende Reagenz in 3- bis 6-mal höherer Konzentration als normalerweise verwendet eingesetzt wird.
  8. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei die Membran zur Verdampfung flüssiger Bestandteile einer Temperatur von 40°C bis 70°C ausgesetzt wird.
  9. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach einem der vorhergehenden Ansprüche, wobei es sich bei der geeigneten Vorrichtung um ein Biolumineszenz-Bildanalysesystem handelt.
  10. Verfahren zur Bestimmung der Anzahl an lebensfähigen Zellen nach Anspruch 9, wobei der Fernsehkamerakopf des Biolumineszenz-Bildanalysesystems entweder sich verjüngende Fasern, eine Photostromverstärkungseinheit und eine Bildröhre oder sich verjüngende Fasern und eine gekühlte CCD-Fernsehkamera umfaßt.
  11. Verfahren zur Bestimmung der Anzahl an lebensfähigen Mikroben nach Anspruch 9 oder 10, wobei das Biolumineszenz-Bildanalysesystem die Anzahl an lebensfähigen Mikroben nach einer Bildverarbeitung derart bestimmt, daß nicht von lebensfähigen Mikroben herrührende Hintergrundlumineszenz ausgelöscht und intensives Licht oberhalb der Schwellenintensität emittierende helle Flecken angezeigt werden.
EP93105182A 1992-04-01 1993-03-29 Verfahren zur Bestimmung der Anzahl lebender Mikroorganismen Expired - Lifetime EP0563858B1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP105299/92 1992-04-01
JP10529992 1992-04-01
JP23420/93 1993-01-20
JP02342093A JP3208892B2 (ja) 1992-04-01 1993-01-20 生菌数の測定方法

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EP0563858A1 EP0563858A1 (de) 1993-10-06
EP0563858B1 true EP0563858B1 (de) 1997-07-09

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EP0563858A1 (de) 1993-10-06
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DE69311944T2 (de) 1997-11-20
DE69311944D1 (de) 1997-08-14

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